Our studies with purified nitric oxide synthase from rat cerebellum are inconsistent with other workers' proposal that tetrahydrobiopterin plays a stoichiometric role in providing reducing equivalents required for the overall oxygenation of arginine to nitric oxide and citrulline. Activity is initially independent of added tetrahydrobiopterin; enhanced product formation with tetrahydrobiopterin is observed only as the incubation progresses. The effect of tetrahydrobiopterin is catalytic, with each mole of added tetrahydrobiopterin supporting the formation of more than 15 moles of product. Recycling of tetrahydrobiopterin was excluded by direct measurement and by the demonstration that nitric oxide synthase is not inhibited by methotrexate (MTX). These combined findings exclude tetrahydrobiopterin as a stoichiometric reactant and suggest that tetrahydrobiopterin enhances product formation by protecting enzyme activity against progressive loss. Preliminary studies indicate that the decreased activity in the absence of added tetrahydrobiopterin does not depend on catalytic turnover of the enzyme. The role of tetrahydrobiopterin may be allosteric or it may function to maintain some group(s) on the enzyme in a reduced state required for activity.